Our long-term goal is the development of tools for characterizing the functions of neuropeptides in neural circuits and non-neuronal tissues in vivo in intact behaving animals. We have developed a new technology, where neuropeptides are transgenically expressed as chimeric fusion proteins tethered to the plasma membrane via hydrophobic anchors ("t-peptides"). T-peptides activate their cognate G-protein coupled receptors (GPCRs) with expected specificity in t-peptide-expressing cells, but without activating their GPCRs in the membranes of neighboring cells not expressing the t-peptide. T- peptides thus provide genetically encoded tools for the cell-autonomous gain-of-function analysis of neuropeptide function in transgenic animals. The Specific Aims are designed to provide a genome-wide toolkit of genetically encoded cell-autonomous pharmacologically specific activators of neuropeptide GPCRs. Because this toolkit will be based on the UAS-GAL4 binary expression system that separates cell- and circuit- specific promoter GAL4 driver transgenes from UAS transgenes containing cDNAs that encode effectors such as t-peptides, it will be of direct utility to Drosophila biologists interested in cell-specific physiological and behavioral functions of neuropeptides. More generally, the development and validation of t-peptide libraries will also provide invaluable insights enabling the generation of tools for use in addressing mammalian neuropeptide function in vivo in health and disease, including potential clinical utility in gene therapy applications. PUBLIC HEALTH RELEVANCE: Bioactive secreted neuropeptides are key regulators of many developmental, physiological, and behavioral processes: metabolism, lifespan, pain sensation, circadian rhythms, sleep, sexual behavior, etc. Human disorders caused by dysfunction of neuropeptide signaling mechanisms-such as narcolepsy, obesity, addiction, post- traumatic stress disorder, intractable pain, etc-are a major source of morbidity, mortality, and economic hardship. Their amelioration will be facilitated by understanding the normal in vivo functions served by the numerous neuropeptides encoded in metazoan genomes.